Methods for producing recombinant glycoproteins with modified glycosylation

ABSTRACT

Genetically engineered host animal cells capable of producing glycoproteins having modified glycosylation patterns, e.g., defucosylation and/or monoglycosylation. Such host animal cells can be engineered to express fucosidase, endoglycosidase or both.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/954,337, filed on Mar. 17, 2014, the content of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Glycosylation is important to the structures and functions ofglycoproteins. For example, glycosylation is suggested to affect proteinfolding (and thus stability) and/or bioactivities of glycoproteins. Thedemand of therapeutic recombinant glycoproteins, especially monoclonalantibodies, robustly grows in the recent two decades. Previous studiesreveal that minor differences in glycan structures of recombinantglycoproteins may impact on the biological activities andpharmacokinetics of the glycoproteins. For example, Darbepoetin alfa isa hyper-glycosylated analog of recombinant human erythropoietin (EPO)with two extra N-linked glycosylation sites. The extra N-glycosylationincreases the percentage of the molecular mass in carbohydrates andsignificantly extends the serum half-life of Darbepoetin alfa, ascompared to endogenous and recombinant EPO. In addition, for atherapeutic antibody whose efficacy mainly relies on antibody-dependentcell cytotoxicity (ADCC), both chemo-enzymatic and genetic approaches toremove the core fucose residue on the Fc portion have been developed toincrease the potency of the ADCC effect induced by that antibody.

However, currently available methods for remodeling glycosylation oftenrequire multiple enzymes and/or multiple steps, resulting in high costsfor manufacturing glyco-engineered recombinant proteins.

SUMMARY OF THE INVENTION

The present disclosure is based on the development of geneticallyengineered host animal cells capable of producing glycoproteins such asantibodies having modified glycosylation, including defucosylation andmonoglycosylation. Such host animal cells were engineered to overlyexpress one or more of fucosidases, endoglycosidases, or both.Unexpectedly, changes to the cellular glycosylation machinery in thehost animal cells did not result in adverse effects in relation toglycoprotein synthesis and host cell growth.

Accordingly, the present disclosure provides a genetically engineeredhost animal cell (e.g., a mammalian cell), which overly expresses afucosidase, an endoglycosidase, or both, wherein the host animal cellproduces glycoproteins having modified glycosylation as compared withthe wild-type couterpart. In some examples, the fucosidase can be amammalian fucosidase or a bacterial fucosidase, for example, humanFUCA1, human FUCA2, Cricetulus griseus fucosidase, alpha-L-1Chryseobacterium meningosepticum α1,6-fucosidase, or bacterialfucosidase BF3242. Alternatively or in addition, the endoglycosidase canbe an Endo S enzyme, e.g., an enzyme comprising the amino acid sequenceof SEQ ID NO:11. In some examples, the genetically engineered hostanimal cell expresses (i) human FUCA1, human FUCA2, Cricetulus griseusfucosidase, alpha-L-1 Chryseobacterium meningosepticum α1,6-fucosidase,or bacterial fucosidase BF3242, and (ii) an Endo S (such as SEQ IDNO:11).

The genetically engineered host animal cell described herein may furtherexpress a glycoprotein, which can be exogenous (not expressed in thenative animal cell of the same type). Examples include, but are notlimited to, an antibody, an Fc-fusion protein, a cytokine, a hormone, agrowth factor, or an enzyme.

In some examples, the genetically engineered host animal cell is amammalian cell, e.g., a Chinese hamster ovary (CHO) cell, a rat myelomacell, a baby hamster kidney (BHK) cell, a hybridoma cell, a Namalwacell, an embryonic stem cell, or a fertilized egg.

Also described herein are methods for producing glycoproteins havingmodified glycosylation patterns (e.g., defucosylated ormono-glycosylated) using any of the genetically engineered host animalcells described herein. The method may comprise (i) providing a hostanimal cell expressing (a) a glycoprotein, and (b) a fucosidase, anendoglycosidase, or both; culturing the host animal cell underconditions allowing for producing the glycoprotein and the fucosidase,the endoglycosidase, or both; (ii) collecting the host animal cell orthe culturing supernatant for isolating the glycoprotein, and optionally(iii) isolating the glycoprotein. The method may further comprise (iv)analyzing the glycosylation pattern of the glycoprotein.

Further, the present disclosure features a method for preparing any ofthe genetically engineered host animal cells described herein. Themethod may comprise (i) introducing into an animal cell one or moreexpression vectors, which collectively encode a fucosidase, anendoglycosidase, or both, and optionally (ii) introducing into theanimal cell an expression vector encoding a glycoprotein. The method mayfurther comprise selecting transformed cells expressing the fucosidase,the endoglycosidase, and the glycoprotein.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration showing the structures of variousN-glycans. A: typical N-linked glycans of glycoproteins. B:defucosylated N-glycans. C: N-glycans having a mono-Nacetylglucosaminereducing sugar (monoglycosylated).

FIG. 2 is a schematic illustration of an exemplary expression vector forproducing a fucosidase. A: map of an exemplary plasmid that carries afucosidase gene. B: an exemplary expression cassette for expressing afucosidase or an endoglycosidase S.

FIG. 3 includes diagrams showing the production of homogenousafucosylated mono-sugar (GlcNAc) antibody h4B12 by transient expressionof the antibody in host cells engineered to express a fucosidase and/oran endoglycosidase. A: a schematic illustration of producing homogenousafucosylated mono-sugar (GlcNAc) antibodies. B: a chart showing theglycosylation of antibody 4B12 produced in host cells engineered toexpress human FUCA1, human FUCA2, C. griseus fucosidase, alpha-L-1, orC. meningosepticum α1,6-fucosidase, as determined by LC/MS/MS. C: adiagram showing the transient expression of a fucosidase or anendoglycosidase as indicated in CHO cells detected by Western blot. D: achart showing antibody h4B12 produced in CHO cells expressing afucodisase or an endoglycosidase as indicated, the antibody havinghomogenous fucose-free mono-sugar (GlcNAc) glycoform as determined byLC/MS/MS. A homogenous N-glycan refers to the ratio of that N-glycan(e.g., an afucosylated N-glycan) to the total N-glycans in aglycoprotein such as an antibody.

FIG. 4 includes diagrams showing the production of homogenousafucosylated mono-sugar (GlcNAc) antibody rituximab by transientexpression of the antibody in host cells engineered to express afucosidase and/or an endoglycosidase. A: a chart showing theglycosylation of antibody rituximab produced in host cells engineered toexpress various fucosidases or endoglycosidases as indicated. B: adiagram showing the transient expression of a fucosidase (left lane) oran endoglycosidase (right lane) as indicated in CHO cells as detected byWesternblot.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are genetically engineered host animal cells such asmammalian cells capable of producing glycoproteins (e.g., exogenousglycoproteins such as antibodies) having modified glycosylation patterns(e.g., modified N-glycosylation patterns such as defucosylated N-glycansor mono-sugar glycans). Such host animal cells may be engineered tooverly express a fucosidase, an endoglycosidase, or both. Optionally,the host animal cell is also engineered to express an exogenousglycoprotein such as an antibody.

The structure of a typical complex N-glycan of glycoproteins produced inwild-type mammalian cells is shown in FIG. 1, panel A. Such a complexN-glycan contains a N-acetylglucosamine (GlcNAc) residue attached to aglycosylation site, an asparagine residue (Asn) of a glycoprotein, and afucose residue is attached to that Asn residue in an alpha1,6-linkage.The genetically engineered host animal cells are capable of producingglycoproteins (e.g., endogenous or exogenous) having modified N-glycans,such as defucosylated N-glycans, an example of which is provided in FIG.1, panel B, and mono-sugar N-glycans, in which only a GlcNAc residue isattached to the Asn glycosylation site (FIG. 1, panel C). A glycoproteinhaving modified glycosylation refers to a glycoprotein carrying at leastone glycan such as an N-glycan that is structurally different fromglycans of the glycoprotein produced in the wild-type counterpart of thegenetically engineered host animal cell. A defucosylated glycan refersto any glycan that does not contain an alpha1,6-fucose residue or anyfucose residue.

A. Fucosidase

A fucosidase is an enzyme that breaks down fucose. This enzyme cleavesfucose residues from a glycan containing such. The fucosidase for use inmaking the genetically engineered host animal cells can be a mammalianfucosidase or a bacterial fucosidase. In some embodiments, thefucosidase is a wild-type enzyme, e.g., a wild-type bacterial enzyme ora wild-type mammalian enzyme such as a human enzyme. The amino acidsequences and encoding nucleotide sequences of a number of exemplaryfucosidases are provided below (including a His-tag at the C-terminus):

Human Fucosidase FUCA1 Amino Acid Sequence (SEQ ID NO: 1)MRVPAQLLGLLLLWLPGARCQPPRRYTPDWPSLDSRPLPAWFDEAKFGVFIHWGVFSVPAWGSEWFWWHWQGEGRPQYQRFMRDNYPPGFSYADFGPQFTARFFHPEEWADLFQAAGAKYVVLTTKHHEGFTNWPSPVSWNWNSKDVGPHRDLVGELGTALRKRNIRYGLYHSLLEWFHPLYLLDKKNGFKTQHFVSAKTMPELYDLVNSYKPDLIWSDGEWECPDTYWNSTNFLSWLYNDSPVKDEVVVNDRWGQNCSCHHGGYYNCEDKFKPQSLPDHKWEMCTSIDKFSWGYRRDMALSDVTEESEIISELVQTVSLGGNYLLNIGPTKDGLIVPIFQERLLAVGKWLSINGEAIYASKPWRVQWEKNTTSVWYTSKGSAVYAIFLHWPENGVLNLESPITTSTTKITMLGIQGDLKWSTDPDKGLFISLPQLPPSAVPAEFAWTIKLTGVKHHHHHH Nucleotide Sequence(SEQ ID NO: 2; codon optimized)atgagagtgcctgctcagctgctgggactgctgctgctgtggctgcctggtgctagatgccagccccctcggagatacacccctgactggccttccctggactccagacctctgcccgcttggtttgacgaggccaagttcggcgtgttcatccactggggcgtgttctccgtgcctgcctggggctctgagtggttctggtggcattggcagggcgagggcagacctcagtaccagcggttcatgcgggacaactacccccctggcttctcctacgccgacttcggccctcagttcaccgcccggttcttccaccctgaggaatgggccgatctgttccaggccgctggcgccaaatacgtggtgctgaccaccaagcaccacgagggcttcaccaactggccctcccccgtgtcctggaactggaactctaaggacgtgggcccccaccgggatctcgtgggagaactgggaaccgccctgcggaagcggaacatcagatacggcctgtaccactccctgctggaatggttccaccccctgtacctgctggacaagaagaacggcttcaagacccagcacttcgtgtccgccaagaccatgcccgagctgtacgacctcgtgaactcctacaagcccgacctgatttggagcgacggcgagtgggagtgccccgacacctattggaactccaccaactttctgtcctggctgtacaacgactcccctgtgaaggacgaggtggtcgtgaacgacagatggggccagaactgctcctgtcaccacggcggctactacaactgcgaggacaagttcaagccccagtccctgcccgaccacaagtgggagatgtgcacctctatcgacaagttctcctggggctaccggcgggacatggccctgtctgatgtgaccgaggaatccgagatcatctccgagctggtgcagaccgtgtccctgggcggcaactacctgctgaacatcggccctaccaaggacggcctgatcgtgcccatcttccaggaacggctgctggccgtgggcaagtggctgtctatcaacggcgaggccatctacgcctccaagccttggcgagtgcagtgggagaagaacaccacctccgtgtggtacacctccaagggctctgccgtgtacgccatcttcctgcactggcccgagaacggcgtgctgaacctggaatcccccatcaccacctctaccaccaagatcaccatgctgggcatccagggcgacctgaagtggtccaccgaccctgacaagggcctgttcatctccctgccccagctgcctccttccgctgtgcctgctgagttcgcctggaccatcaagctgaccggcgtgaagcaccaccaccatcac cattgaHuman Fucosidase FUCA2: Amino Acid Sequence (SEQ ID NO: 3)MRVPAQLLGLLLLWLPGARCHSATRFDPTWESLDARQLPAWFDQAKFGIFIHWGVFSVPSFGSEWFWWYWQKEKIPKYVEFMKDNYPPSFKYEDEGPLFTAKFFNANQWADIFQASGAKYIVLTSKHHEGFTLWGSEYSWNWNAIDEGPKRDIVKELEVAIRNRTDLRFGLYYSLFEWFHPLFLEDESSSFHKRQFPVSKTLPELYELVNNYQPEVLWSDGDGGAPDQYWNSTGFLAWLYNESPVRGTVVTNDRWGAGSICKHGGFYTCSDRYNPGHLLPHKWENCMTIDKLSWGYRREAGISDYLTIEELVKQLVETVSCGGNLLMNIGPTLDGTISVVFEERLRQMGSWLKVNGEAIYETHTWRSQNDTVTPDVWYTSKPKEKLVYAIFLKWPTSGQLFLGHPKAILGATEVKLLGHGQPLNWISLEQNGIMVELPQLTIHQMPCKWGWALALTNVIHHHHHH Nucleotide Sequence(SEQ ID NO: 4; codon optimized)atgagagtgcctgctcagctgctgggactgctgctgctgtggctgcctggcgctagatgccactccgccaccagattcgaccccacctgggagtctctggacgccagacagctgcccgcttggtttgaccaggccaagttcggcatcttcatccactggggcgtgttctccgtgcccagcttcggctctgagtggttctggtggtactggcagaaagagaagatccccaaatacgtggagttcatgaaggacaactacccccccagctttaagtacgaggacttcggccccctgttcaccgccaagttcttcaacgccaaccagtgggccgacatcttccaggcctctggcgccaagtacatcgtgctgacctccaagcaccacgagggcttcaccctgtggggctccgagtactcctggaactggaacgccatcgacgagggccccaagcgggacatcgtgaaagaactggaagtggccatccggaaccggaccgacctgagattcggcctgtactactccctgttcgagtggttccaccccctgtttctggaagatgagtcctccagcttccacaagcggcagttccccgtgtccaagaccctgcccgagctgtacgagctcgtgaacaactaccagcccgaggtgctgtggagtgacggggatggtggtgcccccgatcagtactggaactctaccggcttcctggcctggctgtacaacgagtctcctgtgcggggcaccgtcgtgaccaacgatagatggggcgctggctccatctgcaagcacggcggcttctacacctgttccgaccggtacaaccccggccatctgctgcctcacaagtgggagaactgcatgaccatcgacaagctgtcctggggctacagaagagaggccggcatctccgactacctgacaatcgaggaactcgtgaagcagctggtggaaaccgtgtcctgcggcggcaacctgctgatgaacatcggccctaccctggacggcaccatctccgtggtgttcgaggaacggctgcggcagatgggctcctggctgaaagtgaacggcgaggccatctacgagacacacacctggcggtcccagaacgacaccgtgacccctgacgtgtggtacaccagcaagcccaaagaaaagctggtgtatgccatcttcctgaagtggcctacctccggccagctgttcctgggccaccctaaggctatcctgggcgccaccgaagtgaaactgctgggccatggacagcccctgaactggatctccctggaacagaacggcatcatggtggaactgccccagctgaccatccatcagatgccctgcaaatggggctgggccctggccctgaccaacgtgatccaccatcaccaccaccactga Cricetulus griseus (Chinese Hamster) Fucosidase FUCA2Amino Acid Sequence (SEQ ID NO: 5)MRVPAQLLGLLLLWLPGARCKSSRRYDPTWESLDRRPLPSWFDQAKEGIFIHWGVFSVPSFGSEWFWWYWQKEKRPKFVDFMNNNYPPGFKYEDFGVLFTAKFFNASQWADILQASGAKYLVLTSKHHEGFTLWGSEYSWNWNAVDEGPKRDIVKELKVAITKNTDLRFGLYYSLFEWFHPLFLEDKLSSFQKRQFPISKMLPELYELVNKYQPDILWTDGDGGAPDRYWNSTGFLAWLYNESPVRNTVVTNDRWGAGSICKHGGYYTCSDRYNPGHLLPHKWENCMTIDQFSWGYRREAVISDYLTIEELVKQLVETVACGGNLLMNIGPTLDGIIPVIFEERLRQMGMWLKVNGEAIYETQPWRSQNDTATPDVWYTYKPEEKIVYAIFLKWPVSRELFLEQPIGSLGETEVALLGEGKPLTWTSLKPNGIIVELPQLTLHQMPCKWGWTLALTNVTHHHHHH Nucleotide Sequence(SEQ ID NO: 6; codon optimized)atgagagtgcctgctcagctgctgggactgctgctgctgtggctgcctggcgctagatgcaagtcctctcggagatacgaccccacctgggagtccctggacagaaggcctctgcccagttggttcgaccaggccaagttcggcatcttcatccactggggcgtgttctccgtgcccagcttcggctctgagtggttctggtggtactggcagaaagagaagcggcccaagttcgtggacttcatgaacaacaactacccccctggctttaagtacgaggacttcggcgtgctgttcaccgccaagttcttcaacgcctcccagtgggccgacatcctgcaggcttccggcgctaagtacctggtgctgacctccaagcaccacgagggctttaccctgtggggctccgagtactcctggaactggaacgccgtggacgagggccctaagcgggacatcgtgaaagaactgaaggtggccatcaccaagaacaccgacctgagattcggcctgtactactccctgttcgagtggttccaccccctgtttctggaagataagctgtccagcttccagaagcggcagttccccatctccaagatgctgcccgagctgtacgagctcgtgaacaagtaccagcctgacatcctgtggaccgacggggatggtggcgcccctgacagatactggaactctaccggcttcctggcctggctgtacaacgagtcccctgtgcggaacaccgtcgtgaccaacgacagatggggcgctggctccatctgcaagcacggcggctactacacctgttccgaccggtacaaccccggccatctgctgcctcacaagtgggagaactgcatgacaatcgaccagttctcctggggctaccggcgcgaggccgtgatctctgactacctgaccatcgaggaactcgtgaagcagctggtggaaaccgtggcctgtggcggcaacctgctgatgaacatcggccctaccctggacggcatcatccccgtgatcttcgaggaacggctgcggcagatgggcatgtggctgaaagtgaacggcgaggccatctacgagacacagccttggcggtcccagaacgacaccgccacacctgacgtgtggtacacctacaagcccgaagagaagatcgtgtacgccatcttcctgaagtggcccgtgtccagagagctgtttctggaacagcccatcggctccctgggcgagacagaagtggctctgctgggcgagggcaagcctctgacctggacctccctgaagcccaatggcatcatcgtggaactgccccagctgaccctgcaccagatgccctgtaaatggggctggaccctggccctgaccaacgtgacccaccaccaccatcaccactga Chryseobacterium meningosepticum α1,6-FucosidaseAmino Acid Sequence (SEQ ID NO: 7)MRVPAQLLGLLLLWLPGARCHNVSEGYEKPADPLVVQNLEQWQDLKFGLFMHWGTYSQWGIVESWSLCPEDESWTQRKPEHGKSYNEYVKNYENLQTTFNPVQFNPQKWADATKKAGMKYVVFTTKHHDGFAMFDTKQSDYKIISSKTPFSKNPKADVAKEIENTERDNGFRIGAYESKPDWHSDDYWWSYFPPKDRNVNYDPQKYPARWENFKKFTENQLNEITSNYGKIDILWLDGGWVRPFHTIDPNIEWQRTIKVEQDIDMDKIGTMARKNQPGIIIVDRTVPGKWENYVTPEQAVPEHALSIPWESCITMGDSFSYVPNDNYKSSQKIIETLIRIISRGGNYLMNIAPGPNGDYDAVVYERLKEISGWMDKNQSAVETTRALAPYHESDFYYTQSKDGKIVNVFHISEKSNYQAPSELSFSIPENINPKTVKVLGISSQIKWKKKGNKIHVQLPEERTKLNYSTVIQITQHHHHH HNucleotide Sequence (SEQ ID NO: 8; codon optimized)atgagagtgcctgctcagctgctgggactgctgctgctgtggctgcctggcgctagatgccacaatgtgtccgagggctacgagaagcccgccgaccctctggtggtgcagaacctggaacagtggcaggacctgaagttcggcctgttcatgcactggggcacctactcccagtggggcatcgtggaatcctggtccctgtgccctgaggacgagtcttggacccagcggaagcctgagcacggcaagtcctacaacgagtacgtgaagaactacgagaacctgcagaccaccttcaaccccgtgcagttcaacccccagaagtgggccgacgccaccaagaaagccggcatgaaatacgtggtgttcaccaccaagcaccacgacggcttcgccatgttcgacaccaagcagtccgactacaagatcacctcctccaagacccccttcagcaagaaccccaaggccgacgtggccaaagagattttcaacaccttccgggacaacggcttccggatcggcgcctacttctccaagcctgactggcactccgacgactactggtggtcctacttcccacccaaggaccggaacgtgaactacgaccctcagaaataccccgccagatgggagaacttcaagaagttcaccttcaatcagctgaacgagatcaccagcaactacggcaagatcgacatcctgtggctggacggcggatgggtgcgacccttccacaccatcgaccccaacatcgagtggcagcggaccatcaaggtggaacaggacatcgacatggacaagatcggcaccatggcccggaagaaccagcccggcatcatcatcgtggaccggaccgtgcctggcaagtgggagaattacgtgacccccgagcaggccgtgcctgagcatgccctgtctatcccttgggagtcctgtatcacaatgggcgacagcttctcctacgtgcccaacgacaactacaagtcctcccagaagatcatcgagacactgatcaggatcatctccagaggcggcaactacctgatgaatatcgcccctggccccaacggcgactacgacgctgtggtgtacgagcggctgaaagaaatctccggctggatggataagaaccagtccgccgtgtttaccacccgggctctggccccttaccacgagtccgacttctactacacccagtccaaggacggaaagatcgtgaacgtgttccacatctccgagaagtccaactaccaggccccctccgagctgtccttcagcatccccgagaacatcaaccccaagaccgtgaaggtgctgggcatctccagccagatcaagtggaagaagaagggcaacaagatccacgtgcagctgcccgaggaacggaccaagctgaactactccaccgtgatccagatcacccagcaccaccaccatcac cactgaBacterial Fucosidase BF3242 Amino Acid Sequence (SEQ ID NO: 9)MRVPAQLLGLLLLWLPGARCQQKYQPTEANLKARSEFQDNKFGIFLHWGLYAMLATGEWTMTNNNLNYKEYAKLAGGFYPSKFDADKWVAAIKASGAKYICFTTRHHEGFSMFDTKYSDYNIVKATPFKRDVVKELADACAKHGIKLHFYYSHIDWYREDAPQGRTGRRTGRPNPKGDWKSYYQFMNNQLTELLTNYGPIGAIWFDGWWDQDINPDFDWELPEQYALIHRLQPACLVGNNHHQTPFAGEDIQIFERDLPGENTAGLSGQSVSHLPLETCETMNGMWGYKITDQNYKSTKTLIHYLVKAAGKDANLLMNIGPQPDGELPEVAVQRLKEVGEWMSKYGETIYGTRGGLVAPHDWGVTTQKGNKLYVHILNLQDKALFLPIVDKKVKKAVVFADKTPVRETKNKEGIVLELAKVPTDVDYVVELTIDHHHHHH Nucleotide Sequence(SEQ ID NO: 10; codon optimized)atgagagtgcctgctcagctgctgggactgctgctgctgtggctgcctggtgctagatgccagcagaagtaccagcccaccgaggccaacctgaaggccagatccgagttccaggacaacaagttcggcatcttcctgcactggggcctgtacgccatgctggctactggcgagtggaccatgaccaacaacaacctgaactacaaagagtacgctaagctggctggcggcttctacccctccaagttcgacgccgacaaatgggtggccgccatcaaggcctctggcgccaagtacatctgcttcaccacccggcaccacgagggcttctccatgttcgacaccaagtactccgactacaacatcgtgaaggccacccccttcaagcgggacgtcgtgaaagagctggccgacgcctgcgctaagcacggcatcaagctgcacttctactactcccacatcgactggtacagagaggacgccccccagggcagaaccggcagaagaacaggcagacccaaccccaagggcgactggaagtcctactaccagtttatgaacaaccagctgaccgagctgctgaccaactacggccccatcggcgccatttggttcgacgggtggtgggaccaggacatcaaccccgacttcgactgggagctgcccgagcagtacgccctgatccacagactgcagcccgcctgtctcgtgggcaacaaccaccaccagaccccctttgccggcgaggacatccagattttcgagcgggatctgcccggcgagaacaccgctggactgtctggccagtccgtgtcccatctgcccctggaaacctgcgagacaatgaacggcatgtggggctacaagatcaccgaccagaactacaagtccaccaagacactgatccactacctcgtgaaagccgctggcaaggacgccaacctgctgatgaacatcggcccccagcctgacggcgagctgcctgaagtggctgtgcagcggctgaaagaagtgggagagtggatgtctaagtacggcgagactatctacggcaccagaggcggcctggtggcccctcatgattggggcgtgaccacccagaagggcaacaagctgtacgtgcacatcctgaacctgcaggacaaggccctgttcctgcccatcgtggacaagaaagtgaagaaagccgtggtgttcgccgacaagacccccgtgcggttcaccaagaacaaagagggcatcgtgctggaactggccaaggtgcccaccgacgtggactacgtggtggaactgaccatcgaccaccatcatcaccaccac tga

In some embodiments, the fucosidase can be an enzyme (e.g., a wild-typeenzyme) that share at least 85% (e.g., 90%, 93%, 95%, 96%, 97%, 98%, or99%) sequence identity as compared with any of the exemplary fucosidasesprovided above (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, or SEQ ID NO:9, as well as other fucosidases described herein).

The “percent identity” of two amino acid sequences is determined usingthe algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad.Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into theNBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol.Biol. 215:403-10, 1990. BLAST protein searches can be performed with theXBLAST program, score=50, wordlength=3 to obtain amino acid sequenceshomologous to the protein molecules of the invention. Where gaps existbetween two sequences, Gapped BLAST can be utilized as described inAltschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. Whenutilizing BLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used.

Mammalian fucosidases that can be used in constructing the geneticallyengineered host animal cells include, but are not limited to, thosedisclosed under GenBank Accession Nos. NP_114409.2, XP_003811598.1,AAH03060.1, EHH53333.1, XP_001127152.1, XP_010360962.1, XP_006084558.1,XP_004263802.1, XP_007171384.1, XP_006075254.1, XP_010982011.1,NP_001004218.1, and XP_010964137.1.

Bacterial fucosidases that can be used in constructing the geneticallyengineered host animal cells include, but are not limited to, thosedisclosed under GenBank Accession Nos. WP_008769537.1, WP_032568292.1,EYA08300.1, WP_005780841.1, EXY26528.1, WP_044654435.1, WP_029425671.1,WP_022470316.1, CDA84816.1, WP_004307183.1, and WP_008025871.1.

B. Endoglycosidase

An Endoglycosidase is an enzyme that breaks the glycosidic bonds betweentwo sugar monomers in a glycan, thereby releasing oligosaccharides fromglycoproteins or glycolipids. Endoglycosidase for use in the presentdisclosure (e.g., a wild-type enzyme) includes endoglycosidase D,endoglycosidase F, endoglycosidase F1, endoglycosidase F2,endoglycosidase H, and endoglycosidase S. Exemplary endoglycosidaseenzymes of each subgenus are provided in the table below:

Entry Entry name Protein names Gene names Organism P36911 EBA1_ELIMEEndo-beta-N-acetylglucosaminidase endOF1 Elizabethkingia F1 (EC3.2.1.96) (Di-N- meningoseptica acetylchitobiosyl beta-N-(Chryseobacterium acetylglucosaminidase F1) meningosepticum)(Endoglycosidase F1) (Mannosyl- glycoprotein endo-beta-N-acetyl-glucosaminidase F1) P36913 EBA3_ELIME Endo-beta-N-acetylglucosaminidaseendOF3 Elizabethkingia F3 (EC 3.2.1.96) (Di-N- meningosepticaacetylchitobiosyl beta-N- (Chryseobacterium acetylglucosaminidase F3)meningosepticum) (Endoglycosidase F3) (Mannosyl- glycoproteinendo-beta-N-acetyl- glucosaminidase F3) P36912 EBA2_ELIMEEndo-beta-N-acetylglucosaminidase endOF2 Elizabethkingia F2 (EC3.2.1.96) (Di-N- meningoseptica acetylchitobiosyl beta-N-(Chryseobacterium acetylglucosaminidase F2) meningosepticum)(Endoglycosidase F2) (Mannosyl- glycoprotein endo-beta-N-acetyl-glucosaminidase F2) P04067 EBAG_STRPL Endo-beta-N-acetylglucosaminidaseStreptomyces H (EC 3.2.1.96) (DI-N- plicatus acetylchitobiosyl beta-N-acetylglucosaminidase H) (Endoglycosidase H) (Endo H)(Mannosyl-glycoprotein endo-beta- N-acetyl-glucosaminidase H) T0JJ04T0JJ04_STRSZ Endoglycosidase (EndoS) (EC M837_00287 Streptococcus equi3.2.1.96) subsp. zooepidemicus SzS31A1

Other suitable endoglycosidase enzymes include those that share at least85% (e.g., 90%, 95%, 98%, or 99%) sequence identity to an of the enzymesdescribed herein. Enzymes having a high sequence homology (e.g., atleast 85% sequence identity) with any of the above-listedendoglycosidase are expected to possess the same biological activity.Such enzymes (e.g., wild-type enzymes) may be retrieved from a genedatabase such as GenBank using one of the above listed enzymes as aquery.

In some embodiments, the endoglycosidase described herein is an Endo Senzyme. Endo S is an endoglycosidase that specifically cleaves N-glycansat the first GlcNAc residues attached to the Asn glycosylation sites ofFc domains in native IgG molecules, resulting in monoglycosylated IgGmolecules, i.e., an IgG molecule having a single GlcNAc attached to anAsn glycosylation site. The amino acid sequence and the encodingnucleotide sequence are provided below:

Endo S Amino Acid Sequence (SEQ ID NO: 11):MRVPAQLLGLLLLWLPGARCAQHDSLIRVKAEDKVVQTSPSVSAIDDLHYLSENSKKEFKEGLSKAGEVPEKLKDILSKAQQADKQAKVLAEMKVPEKIAMKPLKGPLYGGYFRTWHDKTSDPAEKDKVNSMGELPKEVDLAFVFHDWTKDYSLFWQELATKHVPTLNKQGTRVIRTIPWRFLAGGDHSGIAEDTQKYPNTPEGNKALAKAIVDEYVYKYNLDGLDVDIERDSIPKVNGKESNENIQRSIAVFEEIGKLIGPKGADKSRLFIMDSTYMADKNPLIERGAPYIDLLLVQVYGIQGEKGDWDPVARKPEKTMEERWESYSKYIRPEQYMVGFSFYEENAGSGNLWYDINERKDDHNPLNSEIAGTRAERYAKWQPKTGGVKGGIFSYAIDRDGVAHQPKKVSDDEKRTNKAIKDITDGIVKSDYKVSKALKKVMENDKSYELIDQKDFPDKALREAVIAQVGSRRGDLERFNGTLRLDNPDIKSLEGLNKLKKLAKLELIGLSQITKLDSSVLPENIKPTKDTLVSVLETYKNDDRKEEAKAIPQVALTISGLTGLKELNLAGFDRDSLAGIDAASLTSLEKVDLSKNKLDLAAGTENRQIFDVMLSTVSNRVGSNEQTVTFDHQKPTGHYPNTYGTTSLRLPVGEGKIDLQSQLLFGTVTNQGTLINSEADYKAYQEQLIAGRRFVDPGYAYKNFAVTYDAYKVRVTDSTLGVTDEKKLSTSKEETYKVEFFSPTNGTKPVHEAKVVVGAEKTMMVNLAAGATVIKSDSHENAKKVFDGAIEYNPLSFSSKISITFEFKEPGLVKYWRFFNDITRKDDYIKEAKLEAFVGHLEDDSKVKDSLEKSTEWVTVSDYSGEAQEFSQPLDNISAKYWRVTVDTKGGRYSSPSLPELQILGYRLPLTHDYKDDDDKEndo S Nucleotide Sequence (SEQ ID NO: 12; codon optimized)atgagagtgcctgctcagctgctgggcctgctgctgctgtggctgcctggtgctagatgcgcccagcacgactccctgatcagagtgaaggccgaggacaaggtggtgcagacctccccttccgtgtccgccatcgacgacctgcactacctgtccgagaactccaagaaagagttcaaagagggcctgtccaaggccggcgaggtgcccgaaaagctgaaggacatcctgagcaaggctcagcaggccgacaagcaggccaaggtgctggccgagatgaaggtgccagagaagatcgccatgaagcccctgaagggccctctgtacggcggctacttcagaacctggcacgacaagacctccgaccccgccgagaaggacaaagtgaactccatgggcgagctgcccaaagaggtggacctggccttcgtgttccacgactggaccaaggactactccctgttctggcaggaactggccaccaagcacgtgcccaccctgaacaagcagggcaccagagtgatccggacaatcccctggcggtttctggctggcggcgaccactctggaatcgccgaggatacccagaagtaccccaacacccccgagggcaacaaggccctggctaaggccatcgtggacgagtacgtgtacaagtacaacctggacggcctggacgtggacatcgagcgggactccatccctaaagtgaacggcaaagagtccaacgagaacatccagcggtctatcgccgtgttcgaggaaatcggcaagctgatcggccccaagggcgccgacaagtcccggctgttcatcatggactccacctacatggccgataagaaccccctgatcgagagaggcgccccttacatcgatctgctgctggtgcaggtgtacggcatccagggcgagaagggcgattgggaccctgtggcccggaagcctgaaaagaccatggaagagagatgggagtcctactccaagtacatccggcccgagcagtatatggtgggattcagcttctacgaggaaaacgccggctccggcaacctgtggtacgacatcaacgagcggaaggacgaccacaaccctctgaactccgagatcgccggcacccgggctgagagatacgctaagtggcagcccaagaccggcggagtgaagggcggcatcttctcctacgccatcgatagggatggcgtggcccaccagcctaagaaggtgtccgacgacgagaagcggaccaacaaggctatcaaggacatcaccgacggcatcgtgaagtccgactacaaggtgtccaaagccctgaagaaagtgatggaaaacgacaagagctacgagctgatcgaccagaaggacttccccgataaggccctgcgcgaggccgtgattgctcaagtgggctccagacggggcgacctggaaagattcaacggcaccctgcggctggacaaccccgacatcaagtccctggaaggcctgaacaaactgaagaagctggccaagctggaactgatcggactgtcccagatcacaaagctggactcctccgtgctgcctgagaacatcaagcccaccaaggacaccctggtgtccgtgctggaaacctacaagaacgacgaccggaaagaggaagccaaggccatccctcaggtggccctgaccatctctggcctgaccggcctgaaagagctgaatctggccggcttcgaccgggattccctggctggaatcgatgccgcctctctgacctccctggaaaaagtggacctgtctaagaacaagctggatctggctgccggcaccgagaaccggcagatcttcgacgtgatgctgtccaccgtgtccaacagagtgggcagcaacgagcagaccgtgaccttcgaccaccagaagcccaccggccactaccctaacacctacggcaccacctccctgagactgcctgtgggcgagggcaagatcgacctgcagtcccagctgctgttcggcaccgtgaccaaccagggcacactgatcaactccgaggccgattacaaggcctaccaggaacagctgatcgctgggcggagattcgtggaccctggctacgcttacaagaacttcgccgtgacctacgatgcctacaaagtgcgcgtgaccgactccaccctgggcgtgacagacgaaaagaagctgagcacctccaaagaagagacatacaaggtggaattcttctcccccaccaatggcaccaagcctgtgcatgaggctaaggtggtcgtgggcgccgagaaaaccatgatggtcaacctggccgctggcgccaccgtgatcaagtctgactctcacgagaatgccaaaaaggtgttcgacggcgccatcgagtacaatcctctgagcttctccagcaagaccagcatcaccttcgagtttaaagaacccggcctcgtgaaatactggcggttcttcaacgatatcacccgcaaggacgactacatcaaagaggctaagctggaagccttcgtgggccatctggaagatgactccaaagtgaaggactctctggaaaagtccaccgagtgggtcaccgtgtctgactactctggcgaggcccaggaattctcccagcccctggacaacatctccgccaagtattggagagtgaccgtggacaccaagggcggacggtacagctctcctagcctgcccgagctgcagatcctgggctacagactgcctctgacccacgactataaggacgacgacgacaaa tga

In some embodiments, an Endo S enzyme described herein can be an enzyme(e.g., a wild-type enzyme) that share at least 85% (e.g., 90%, 93%, 95%,96%, 97%, 98%, or 99%) sequence identity as compared with SEQ ID NO:11.Examples include, but are not limited to, those described under GenBankAccession Nos. EQB24254.1, WP_037584019.1, WP_012679043.1, andADC53484.1.

C. Genetically Engineered Host Animal Cells

The host animal cells described herein are genetically engineered tooverly express one or more enzymes having specific glycan-modifyingactivities (e.g., glycosidase or glycol-transferase). A geneticallyengineered host animal cell is an animal cell that carry exogenous(non-native) genetic materials, such as exogenous genes encoding one ormore of the fucosidase and endoglycosidase described herein. A host cellthat overly expresses an enzyme refers to a genetically engineered hostcell that expresses the enzyme in a level greater (e.g., 20%, 50%, 80%,100%, 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10⁴-fold,or 10⁵-fold higher) than that of the enzyme in the wild-type counterpartof the host cell, i.e., the same type of cell that does not contain thesame genetic modification as the genetically engineered host cell. Insome embodiments, a gene encoding an exogenous enzyme as describedherein can be introduced into a suitable parent animal cell to producethe genetically engineered host animal cell described herein. Anexogenous enzyme refers to an enzyme that does not exist in the parentcell used for making the engineered host animal cell.

Genetically engineered host animal cells as described herein, which arecapable of producing glycoproteins having modified glycosylation ascompared with the wild-type counterpart, can be prepared by the routinerecombinant technology. In some instances, a strong promoter can beinserted upstream to an endogenous fucosidase and/or endoglycosidasegene to enhance its expression. In other instances, exogenous geneticmaterials encoding one or more of fucosidases and/or endoglycosidasescan be introduced into a parent host cell to produce the geneticallyengineered host animal cells as described herein.

A gene encoding a fucosidase or endoglycosidase as described herein canbe inserted into a suitable expression vector (e.g., a viral vector or anon-viral vector) using methods well known in the art. Sambrook et al.,Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press. For example, the gene and the vector can be contacted,under suitable conditions, with a restriction enzyme to createcomplementary ends on each molecule that can pair with each other and bejoined together with a ligase. Alternatively, synthetic nucleic acidlinkers can be ligated to the termini of a gene. These synthetic linkerscontain nucleic acid sequences that correspond to a particularrestriction site in the vector. In some embodiments, the gene of thefucosidase or endoglycosidase is contained in an expression cassettecomprising one of more of the following elements: a Kozak sequence and asignal peptide sequence, which are located at the N-terminus of theenzyme, and a protein tag (e.g., FLAG, His-tag, include chitin bindingprotein (CBP), maltose binding protein (MBP), andglutathione-S-transferase (GST)). The protein tag can be located ateither the N-terminus or C-terminus of the enzyme. See, e.g., FIG. 2,panel B.

Additionally, the expression vector can contain, for example, some orall of the following: a selectable marker gene, such as the neomycingene for selection of stable or transient transfectants in mammaliancells; enhancer/promoter sequences from the immediate early gene ofhuman CMV for high levels of transcription; transcription terminationand RNA processing signals from SV40 for mRNA stability; SV40 polyomaorigins of replication and ColE1 for proper episomal replication;versatile multiple cloning sites; and T7 and SP6 RNA promoters for invitro transcription of sense and antisense RNA. Suitable vectors andmethods for producing vectors containing transgenes are well known andavailable in the art. Sambrook et al., Molecular Cloning, A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory Press.

If two or more enzymes are to be used in constructing the host animalcells described herein, for example, two or more fucosidases, two ormore endoglycosidases, or a combination of fucosidase andendoglycosidase, genes encoding the two or more enzymes can be insertedinto separate express vectors or inserted into a common express vectordesigned for producing multiple proteins.

Expression vectors for producing the fucosidase and/or endoglycosidasemay be introduced into suitable parent host cells, including, but arenot limited to, murine myeloma cells (e.g., NSO cells), Chinese HamsterOvary (CHO) cells, human embryonic kidney cells (e.g., HEK293), andhuman retinoblastoma cells (e.g., PER.C6). Selection of a suitable hostcell line, which is within the knowledge of those skilled in the art,would depend on the balance between the need for high productivity andthe need for producing the product having desired properties. In someinstances, the expression vectors can be designed such that they canincorporate into the genome of cells by homologous or non-homologousrecombination by methods known in the art. Methods for transferringexpression vectors into the parent host cells include, but are notlimited to, viral mediated gene transfer, liposome mediated transfer,transformation, transfection and transduction, e.g., viral mediated genetransfer such as the use of vectors based on DNA viruses such asadenovirus, adeno-associated virus and herpes virus, as well asretroviral based vectors. Examples of modes of gene transfer includee.g., naked DNA, CaPO₄ precipitation, DEAE dextran, electroporation,protoplast fusion, lipofection, cell microinjection, and viral vectors,adjuvant-assisted DNA, gene gun, catheters. In one example, a viralvector is used. To enhance delivery of non-viral vectors to a cell, thenucleic acid or protein can be conjugated to antibodies or bindingfragments thereof which bind cell surface antigens. Liposomes that alsoinclude a targeting antibody or fragment thereof can be used in themethods described herein.

A “viral vector” as described herein refers to a recombinantly producedvirus or viral particle that comprises a polynucleotide to be deliveredinto a host cell, either in vivo, ex vivo or in vitro. Examples of viralvectors include retroviral vectors such as lentiviral vectors,adenovirus vectors, adeno-associated virus vectors and the like. Inaspects where gene transfer is mediated by a retroviral vector, a vectorconstruct refers to the polynucleotide comprising the retroviral genomeor part thereof, and a therapeutic gene.

The genetically engineered animal host cells can comprise the use of anexpression cassette created for either constitutive or inducibleexpression of the introduced gene(s). Such an expression cassette caninclude regulatory elements such as a promoter, an initiation codon, astop codon, and a polyadenylation signal. The elements can be operablylinked to the gene encoding the surface protein of interest such thatthe gene is operational (e.g., is expressed) in the host cells.

A variety of promoters can be used for expression of the fucosidaseand/or endoglycosidase (as well as any exogenous glycoproteins asdescribed herein). Promoters that can be used to express the protein arewell known in the art, including, but not limited to, cytomegalovirus(CMV) intermediate early promoter, a viral LTR such as the Rous sarcomavirus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) earlypromoter, E. coli lac UV5 promoter and the herpes simplex tk viruspromoter.

Regulatable promoters can also be used. Such regulatable promotersinclude those using the tetracycline repressor (tetR) [Gossen, M., andBujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. etal., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc.Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506dimer, VP16 or p65 using astradiol, RU486, diphenol murislerone orrapamycin. Inducible systems are available from Invitrogen, Clontech andAriad.

The effectiveness of some inducible promoters can be increased overtime. In such cases one can enhance the effectiveness of such systems byinserting multiple repressors in tandem, e.g., TetR linked to a TetR byan internal ribosome entry site (IRES). Alternatively, one can wait atleast 3 days before screening for the desired function. While somesilencing may occur, it can be minimized by using a suitable number ofcells, preferably at least 1×10⁴, more preferably at least 1×10⁵, stillmore preferably at least 1×10⁶, and even more preferably at least 1×10⁷.One can enhance expression of desired proteins by known means to enhancethe effectiveness of this system. For example, using the WoodchuckHepatitis Virus Posttranscriptional Regulatory Element (WPRE). See Loeb,V. E., et al., Human Gene Therapy 10:2295-2305 (1999); Zufferey, R., etal., J. of Virol. 73:2886-2892 (1999); Donello, J. E., et al., J. ofVirol. 72:5085-5092 (1998).

Examples of polyadenylation signals useful to practice the methodsdescribed herein include, but are not limited to, human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

The exogenous genetic material that includes fucosidase gene and/orendoglycosidase gene (as well as a glycoprotein gene as describedherein) operably linked to the regulatory elements may remain present inthe cell as a functioning cytoplasmic molecule, a functioning episomalmolecule or it may integrate into the cell's chromosomal DNA. Exogenousgenetic material may be introduced into cells where it remains asseparate genetic material in the form of a plasmid. Alternatively,linear DNA, which can integrate into the chromosome, may be introducedinto the cell. When introducing DNA into the cell, reagents, whichpromote DNA integration into chromosomes, may be added. DNA sequences,which are useful to promote integration, may also be included in the DNAmolecule. Alternatively, RNA may be introduced into the cell.

Selectable markers can be used to monitor uptake of the desiredtransgene into the host animal cells described herein. These markergenes can be under the control of any promoter or an inducible promoter.These are known in the art and include genes that change the sensitivityof a cell to a stimulus such as a nutrient, an antibiotic, etc. Genesinclude those for neo, puro, tk, multiple drug resistance (MDR), etc.Other genes express proteins that can readily be screened for such asgreen fluorescent protein (GFP), blue fluorescent protein (BFP),luciferase, and LacZ.

D. Producing Glycoproteins Having Modified Glycosylation

The genetically engineered host animal cells can be used for producingglycoproteins (e.g., endogenous or exogenous) having modifiedglycosylation patterns. In some embodiments, the parent host cell foruse to producing the engineered host animal cells described abovealready carries a gene(s) encoding an exogenous glycoprotein. In otherembodiments, a gene or multiple genes encoding a glycoprotein ofinterest can be introduced into the genetically engineered host animalcells that express one or more fucosidase and/or endoglycosidase bymethods known in the art or described herein.

Genetically engineered host animal cells capable of producing both aglycoprotein of interest and one or more of fucosidases and/orendoglycosidases can be cultured under suitable conditions allowing forexpression of these proteins. The cells and/or the culture medium can becollected and the glycoprotein of interested can be isolated andpurified from the cells and/or the culture medium by routine technology.The glycosylation pattern of the glycoprotein thus produced can bedetermined by routine technology, e.g., LC/MS/MS, to confirmmodification of glycosylation.

In some examples, the glycoprotein of interest is an antibody. Exemplaryantibodies include, but are not limited to, abciximab (glycoproteinIIb/IIIa; cardiovascular disease), adalimumab (TNF-α, variousauto-immune disorders, e.g., rheumatoid arthritis), alemtuzumab (CD52;chronic lymphocytic leukemia), basiliximab (IL-2Rα receptor (CD25);transplant rejection), bevacizumab (vascular endothelial growth factorA; various cancers, e.g., colorectal cancer, non-small cell lung cancer,glioblastoma, kidney cancer; wet age-related macular degeneration),catumaxomab, cetuximab (EGF receptor, various cancers, e.g., colorectalcancer, head and neck cancer), certolizumab (e.g., certolizumab pegol)(TNF alpha; Crohn's disease, rheumatoid arthritis), Daclizumab (IL-2Rareceptor (CD25); transplant rejection), eculizumab (complement proteinC5; paroxysmal nocturnal hemoglobinuria), efalizumab (CD11a; psoriasis),gemtuzumab (CD33; acute myelogenous leukemia (e.g., withcalicheamicin)), ibritumomab tiuxetan (CD20; Non-Hodgkin lymphoma (e.g.,with yttrium-90 or indium-111)), infliximab (TNF alpha; variousautoimmune disorders, e.g., rheumatoid arthritis) Muromonab-CD3 (T CellCD3 receptor; transplant rejection), natalizumab (alpha-4 (α4) integrin;multiple sclerosis, Crohn's disease), omalizumab (IgE; allergy-relatedasthma), palivizumab (epitope of RSV F protein; Respiratory SyncytialVirus infection), panitumumab (EGF receptor; cancer, e.g., colorectalcancer), ranibizumab (vascular endothelial growth factor A; wetage-related macular degeneration), rituximab (CD20; Non-Hodgkinlymphoma), tositumomab (CD20; Non-Hodgkin lymphoma), trastuzumab (ErbB2;breast cancer).

In some examples, the glycoprotein of interest is a cytokine. Examplesinclude, but are not limited to, interferons (e.g., IFN-α, INF-β, orINF-γ), interleukins (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12),and colony stimulating factors (e.g., G-CSF, GM-CSF, M-CSF). The IFN canbe, e.g., interferon alpha 2a or interferon alpha 2b. See, e.g., Mott HR and Campbell I D. “Four-helix bundle growth factors and theirreceptors: protein protein interactions.” Curr Opin Struct Biol. 1995February; 5(1):114-21; Chaiken I M, Williams W V. “Identifying structurefunction relationships in four-helix bundle cytokines: towards de novomimetics design.” Trends Biotechnol. 1996 October; 14(10):369-75; KlausW, et al., “The three-dimensional high resolution structure of humaninterferon alpha-2a determined by heteronuclear NMR spectroscopy insolution”. J. Mol Biol., 274(4):661-75, 1997, for further discussion ofcertain of these cytokines.

The protein of interest may also be a cytokine protein that has asimilar structure to one or more of the afore-mentioned cytokines. Forexample, the cytokine can be an IL-6 class cytokine such as leukemiainhibitory factor (LIF) or oncostatin M. In some embodiments, thecytokine is one that in nature binds to a receptor that comprises aGP130 signal transducing subunit. Other four-helix bundle proteins ofinterest include growth hormone (GH), prolactin (PRL), and placentallactogen. In some embodiments, the target protein is an erythropoiesisstimulating agent, e.g., (EPO), which is also a four-helix bundlecytokine. In some embodiments, an erythropoiesis stimulating agent is anEPO variant, e.g., darbepoetin alfa, also termed novel erythropoiesisstimulating protein (NESP), which is engineered to contain five N-linkedcarbohydrate chains (two more than recombinant HuEPO). In someembodiments, the protein comprises five helices. For example, theprotein can be an interferon beta, e.g., interferon beta-1a orinterferon beta-1b, which (as will be appreciated) is often classifiedas a four-helix bundle cytokine. In some embodiments, a target proteinis IL-9, IL-10, IL-11, IL-13, or IL-15. See, e.g., Hunter, C A, NatureReviews Immunology 5, 521-531, 2005, for discussion of certaincytokines. See also Paul, W E (ed.), Fundamental Immunology, LippincottWilliams & Wilkins; 6th ed., 2008.

In addition, the protein of interest may be a protein that is approvedby the US Food & Drug Administration (or an equivalent regulatoryauthority such as the European Medicines Evaluation Agency) for use intreating a disease or disorder in humans. Such proteins may or may notbe one for which a PEGylated version has been tested in clinical trialsand/or has been approved for marketing. In some instances, the proteinof interest is an Fc-fusion protein, including, but not limited to,abatacept, entanercept, IL-2-Fc fusion protein, CD80-Fc fusion protein,and PDL1-Fc fusion protein.

Further, the protein of interest may be a neurotrophic factor, i.e., afactor that promotes survival, development and/or function of neurallineage cells (which term as used herein includes neural progenitorcells, neurons, and glial cells, e.g., astrocytes, oligodendrocytes,microglia). For example, in some embodiments, the target protein is afactor that promotes neurite outgrowth. In some embodiments, the proteinis ciliary neurotrophic factor (CNTF; a four-helix bundle protein) or ananalog thereof such as Axokine, which is a modified version of humanCiliary neurotrophic factor with a 15 amino acid truncation of the Cterminus and two amino acid substitutions, which is three to five timesmore potent than CNTF in in vitro and in vivo assays and has improvedstability properties.

Alternatively, the protein of interest can be an enzyme, e.g., an enzymethat is important in metabolism or other physiological processes. As isknown in the art, deficiencies of enzymes or other proteins can lead toa variety of disease. Such diseases include diseases associated withdefects in carbohydrate metabolism, amino acid metabolism, organic acidmetabolism, porphyrin metabolism, purine or pyrimidine metabolism,lysosomal storage disorders, blood clotting, etc. Examples include Fabrydisease, Gaucher disease, Pompe disease, adenosine deaminase deficiency,asparaginase deficiency, porphyria, hemophilia, and hereditaryangioedema. In some embodiments, a protein is a clotting or coagulationfactor, (e.g., factor VII, VIIa, VIII or IX). In other embodiments aprotein is an enzyme that plays a role in carbohydrate metabolism, aminoacid metabolism, organic acid metabolism, porphyrin metabolism, purineor pyrimidine metabolism, and/or lysosomal storage, wherein exogenousadministration of the enzyme at least in part alleviates the disease.

Further, the protein of interest can be a hormone, such as insulin,growth hormone, Luteinizing hormone, follicle-stimulating hormone, andthyroid-stimulating hormone. The protein of interest can also be agrowth factor, including, but not limited to, adrenomedullin (AM),angiopoietin (Ang), autocrine motility factor, bone morphogeneticproteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermalgrowth factor (EGF), erythropoietin (EPO) fibroblast growth factor(FGF), glial cell line-derived neurotrophic factor (GDNF), granulocytecolony-stimulating factor (G-CSF), granulocyte macrophagecolony-stimulating factor (GM-CSF), growth differentiation factor-9(GDF9), healing factor, hepatocyte growth factor (HGF) hepatoma-derivedgrowth factor (HDGF), insulin-like growth factor (IGF), keratinocytegrowth factor (KGF), migration-stimulating factor (MSF), myostatin(GDF-8), nerve growth factor (NGF) and other neurotrophins,platelet-derived growth factor (PDGF), thrombopoietin (TPO),transforming growth factor alpha(TGF-α), transforming growth factorbeta(TGF-β), tumor necrosis factor-alpha(TNF-α), vascular endothelialgrowth factor (VEGF), and placental growth factor (PGF).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Examples Methods

i. Construction of Expression Vectors for Producing a Fucosidase or anEndoglycosidase.

In order to construct expression vectors for a fucosidase or anendoglycosidase, fucosidase or endoglycosidase gene was isolated byroutine technology and subjected to codon optimization based on codonusage of hamster cells. The synthetic genes were prepared by GeneArtCorp. and cloned into pcDNA3.1 B(−) Myc-His vector (Invitrogen, US) atrestriction sites Bgl II/EcoR I. (FIG. 2, panel A). The expressioncassette of an alpha-fucosidase comprises, from 5′ to 3′, a Kozaksequence, an Igk leader sequence, the coding sequence of the fucosidase,and a sequence encoding a His-tag. FIG. 2, panel B. The expressioncassette of an endoglycosidase comprises, from 5′ to 3′, a Kozaksequence, an Igk leader sequence, the coding sequence of theendoglycosidase, and a sequence encoding a Flag tag. FIG. 2, panel B.

ii. Preparation of Defucosylated Antibody

An antibody producing cell line was maintained at 0.3-3.0×10⁶ viablecells/mL in a complete medium, CD FortiCHO™ medium supplemented with 8mM L-glutamine and anti-Clumping Agent at 1:100 dilution (LifeTechnologies, USA). Cells were maintained on a shaking platform settingat 130-150 rpm in an 8% CO₂ incubator.

To produce defucosylated antibodies, the antibody-producing cells notedabove were transfected with the expression vector encoding analpha-fucosidase described above by FreeStyleMAX reagent (LifeTechnologies, USA) according to manufacturer's protocol. Transfectedcells were cultured in a medium comprising 4 g/L of glucose and themedium was changed every other day. The cells were harvested when thecell viability was dropped below 70% Clarified culture supernatant wascollected and purified by Protein A Chromatography.

iii. Analysis of Glycosylation of Antibodies

Recombinant antibodies prepared according to the methods describedherein were reduced, alkylated, and digested overnight with trypsin inthe presence of 25 mM ammonium bicarbonate buffer (pH˜8) at 37° C.PNGase F solution (3 μL, Roche) was added to 200 μL of the digestedsample and the mixture was incubated for another 16 hours at 37° C. Thereleased glycans were separated from the peptides using a Sep-Pak® C18cartridge (Waters). The Sep-Pak C18 was washed with acetonitrile,followed by water. The PNGaseF digested sample was loaded onto thecartridge and the released glycans were eluted with 1% ethanol while thepeptides remained bound to the Sep-Pak C18. The released proteinoligosaccharides were first purified using a porous graphite carboncolumn (PhyNexus) and then permethylated. All mass spectrometryexperiments were performed using an Orbitrap Fusion Tribrid massspectrometer via direct infusion into the nano-electrospray source.

Results

1. Production of Antibodies h4B12, Rituximab, and Omalizumab HavingMono-Sugar (GlcNAc) Glycoform

A monosaccharide glycovariant could be made from the aforementioneddi-sugar variant by a fucosidase cleavage reaction. Search from a numberof available enzymes and glycol-peptide analysis by LC/MS/MS indicatedthat, with optimized cleavage reaction conditions, an efficientde-fucosidation could be achieved using an α-1,6-fucosidase, and that ahigher cleavage efficiency is associated with a lower NF/N ratio.Alternatively, a mono-sugar glycovariant could be obtained with tworeaction enzymes combined in sequence, including an endoglycosisase(Endo S) and an α-1,6-fucosidase. The resultant mono-GlcNAc glycovariantwas shown in FIG. 3, panel A.

The results show that Endo-S removed >90% N-linked glycans of the heavychain of h4B12, rituximab, and omalizumab produced in the engineered CHOcells described herein. The defucosylation ability of five differenttypes of fucosidases: FUCA1, FUCA2, Cricetulus griseus fucosidase,alpha-L-1, Chryseobacterium meningosepticum α1,6-Fucosidas, and BF3242are 5.8%, 9.1%, 17.7%, 11.5 and 68%, respectively, in relation to h4B12antibodies produced in the CHO cells expressing each of the fucosidase.FIG. 3, panel B. The expression of the enzymes was detect by Westernblot as shown in FIG. 3, panel C.

2-deoxy-2-fluoro L-Fucose is a fluorinated fucose analog. It can bemetabolized inside host cells to generate a substrate-based inhibitor offucosyltransferases. When culturing antibody-producing CHO cellstransiently expressing fucosidase BF3242 and Endo-S, 99.89% of theN-glycans linked to the antibody produced in the CHO cells aremonoglycosylated (GlcNAc-Ig-Fc). FIG. 3, panel D.

CHO-35D6 cells, which produce rituximab, were stable transfected with anexpression vector for producing BF3242 and Endo-S. The cells werecultured in the presence of 100-400 μg/ml G418. The antibody thusproduced contains 17-19% of GlcNAc-Ig-Fc. FIG. 4, panel A. Enzymeexpression was detect by Western blot as shown in FIG. 4, panel B.

Similar results were observed in omalizumab produced in CHO cellsengineered to express both a fucosidase and Endo S.

Such efficiency represents an important step for transglycosylation inthe preparation of antibodies with homogeneous glycan form.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A genetically engineered host animal cell, whichexpresses a fucosidase, an endoglycosidase, or both, wherein theengineered host animal cell produces glycoproteins having modifiedglysocylation.
 2. The genetically engineered host animal cell of claim1, wherein the fucosidase is a mammalian fucosidase or a bacterialfucosidase.
 3. The genetically engineered host animal cell of claim 1,wherein the endoglycosidase is an Endo S enzyme.
 4. The geneticallyengineered host animal cell of claim 1, which further expresses aglycoprotein.
 5. The genetically engineered host animal cell of claim 4,wherein the glycoprotein is exogenous.
 6. The genetically engineeredhost animal cell of claim 5, wherein the glycoprotein is an antibody, anFc-fusion protein, a cytokine, a hormone, a growth factor, or an enzyme.7. The genetically engineered host animal cell of claim 6, wherein theglycoprotein is an antibody.
 8. The genetically engineered host animalcell of claim 1, wherein the animal cell is a mammalian cell.
 9. Thegenetically engineered host animal cell of claim 8, wherein themammalian cell is a Chinese hamster ovary (CHO) cell, a rat myelomacell, a baby hamster kidney (BHK) cell, a hybridoma cell, a Namalwacell, an embryonic stem cell, or a fertilized egg.
 10. The geneticallyengineered host animal cell of claim 1, which expresses both afucosidase and an endoglycosidase.
 11. The genetically engineered hostanimal cell of claim 10, which expresses (i) human FUCA1, human FUCA2,Cricetulus griseus fucosidase, alpha-L-1 Chryseobacteriummeningosepticum α1,6-fucosidase, or bacterial fucosidase BF3242, and(ii) an Endo S.
 12. A method for producing a defucosylated glycoprotein,comprising: providing a genetically engineered host animal cellexpressing (a) a glycoprotein, and (b) a fucosidase, an endoglycosidase,or both; culturing the host animal cell under conditions allowing forproducing the glycoprotein and the fucosidase, the endoglycosidase, orboth; and collecting the host animal cell or the culture supernatant forisolating the glycoprotein.
 13. The method of claim 12, wherein thefucosidase is a mammalian fucosidase or a bacterial fucosidase.
 14. Themethod of claim 12, wherein the endoglycosidase is an Endo S enzyme. 15.The method of claim 12, wherein the glycoprotein is exogenous.
 16. Themethod of claim 12, wherein the glycoprotein is an antibody, anFc-fusion protein, a cytokine, a hormone, a growth factor, or an enzyme.17. The method of claim 16, wherein the glycoprotein is an antibody. 18.The method of claim 12, wherein the genetically engineered host animalcell is a mammalian cell.
 19. The method of claim 18, wherein themammalian cell is a Chinese hamster ovary (CHO) cell, a rat myelomacell, a baby hamster kidney (BHK) cell, a hybridoma cell, a Namalwacell, an embryonic stem cell, or a fertilized egg.
 20. The method ofclaim 12, wherein the genetically engineered host animal cell expressesboth a fucosidase and an endoglycosidase.
 21. The method of claim 12,further comprising isolating the glycoprotein.
 22. The method of claim21, further comprising analyzing the glycosylation pattern of theglycoprotein.
 23. A method for preparing the genetically engineered hostanimal cell of claim 1, comprising introducing into an animal cell oneor more expression vectors, which collectively encode a fucosidase, anendoglycosidase, or both.
 24. The method of claim 23, further comprisingintroducing into the animal cell an expression vector encoding aglycoprotein.